Non-ribosomal peptide synthetase (referred as ‘NRPS’ hereinafter) is organized by at least one ORF (open reading frame) forming NRPS complex, and each NRPS or NRPS subunit comprises one or more modules. A module is defined as the catalystic unit that incorporates one building block (for example, one amino acid) into the growing chain. Order and specificity of the modules within the NRPS determine the sequence and structure of the peptide product. Thus, NRPS which is not involved in ribosomal RNA translation used to be carried out by genetic code can produce peptides of wider structural diversity than those peptides translated from RNA template by ribosome. The peptides produced by NRPS can be further modified by the connection between hydroxyl acid and D- and L-amino acid, mutation and oxidation in main peptide chain forming linear, cyclic or branched cyclic structure, acylation, glycosylation, N-methylation and heterocyclic ring formation.
Polymyxin synthetase, one of NRPSs, stepwisely combines each amino acid monomer forming polymyxin and if necessary transforms the amino acid to complete the entire amino acid chain and to form a ring structure in order to synthesize a peptide antibiotic. Each module of NRPS is organized by at least three domains, which are A, C, and T domains. A domain (adenylation domain) plays a role in the selection and activation of an amino acid monomer, C domain (condensation domain) catalyzes peptide bond formation and T domains (thiolation domain, also called PCP) is involved in rotating phosphopantheteine group to incorporate an amino acid monomer into the growing peptide chain.
Recently, the tertiary structure of A domain recognizing phenylalanine of gramicidin biosynthesis gene has been identified, in which a specific amino acid binding site contains 8 amino acid residues (Conti E. et al., 1997. EMBO J. 16: 4174-4183). The amino acid sequence of this A domain was compared with that of the conventional A domain, as a result this A domain had high homology in 8 amino acid residues with the conventional A domain. Thus, analyzing the 8 amino acid residues may lead to the understanding of the association of a specific A domain with an amino acid (Challis G. L. et al., 2000. Chem. Biol. 7: 211-224).
In addition to these major domains, there are E domain (epimerization domain) playing a role in conversion of L-amino acid into D-amino acid and TE domain (termination domain), which are characterized by a specific amino acid motif.
A novel enzyme characterized by specificity can be designed by the modification of numbers and locations of modules at DNA level by genetic engineering and in vivo recombination techniques. For example, a domain originated from heterologous NRPS is substituted by using a recombinant technique (Schneider et al., Mol. Gen. Genet., 257, pp. 308-318, 1998) or a module can be designed to have specificity by changing residues forming the substrate binding pocket of A domain (Cane et al., Chem. Biol. vol. 6, p. 319-325, 1999).
Unlike other general peptides ribosomally translated, polymyxin is an antibiotic isolated from Bacillus sp. or Paenibacillus sp., which is produced by non-ribosomal peptide synthetase (Marahiel M. A. et al., 1997, Chem. Rev. 97, 2651-2673; Doekel S. et al., 2001, Metab. Eng. 6, 64-77).
The molecular weight of polymyxin is approximately 1200 Da (1.2 kDa) (Storm D. R. et al., Ann Rev. Biochem. 1977; 46:723-763). The basic structure of polymyxin is polyketidic peptide ring comprising 8˜10 amino acids and 2,4-diaminobutyric acid (Dab) at high concentration. Fatty acid is also attached on the peptide ring, which is preferably 6-methyoctanoic acid or 6-methylheptanoic acid (see FIG. 4). This structure favors solubility of polymyxin, suggesting that polymyxin is soluble in both water and an organic solvent.
Polymyxin is an antibiotic that is able to induce apoptosis by changing permeability of cell membrane and is functioning according to the following mechanisms.
First, polyketidic peptide ring of polymyxin changes the bridge between magnesium and calcium that stabilizes lipopolysaccharide of cell membrane to be bound to the cell. Then, lipopolysaccharide of cell membrane is reacted with fatty acid residue of polymyxin to make the linkage between polymyxin and cell membrane strong and tight. At last, polymyxin is incorporated into the outer membrane of cell, resulting in the destruction of the cell membrane (Hermsen E. D. et al., 2003, Infect. Dis. Clin. N. Am. 17: 545-562).
Polymyxin B was first isolated from Paenibacillus polymyxa in 1947 and since then 15 polymyxins have been reported (Storm D. R., et al., 1977, Annu. Rev. Biochem., 46: 723-763; Silaev, A. B. et al., 1975, Zh. Obshch. Khim. 45: 2331-2337; Martin N. I. et al., 2003, J. Biol. Chem. 278: 13124-13132). The polymyxin based antibiotic ‘polymyxin B sulfate’ killed 88% of Pseudomonas aeruginosa at the concentration of 0.01 μg/ml. Polymyxin E showed lethal effect at the concentration of 0.1 μg/ml. Polymyxin B and polymyxin E exhibited lethal effect on most Escherichia coli strains and Pseudomonas aeruginosa at the concentration under 2 μg/ml, in addition to on every Enterobacter, Salmonella, Shigella, Pasteurella, Brucella and Bordetella. However, both polymyxin B and E showed no lethal effect on Proteus, Serratia, Providencia and Edwardsiella even at the higher concentrations than 200 μg/ml. They had no effect on gram-positive bacteria, fungi and anaerobic bacteria, either (Nord N. M. et al., 1964, N. Engl. J. Med. 270: p. 1030-1035).
Thus, polymyxin had been used as a therapeutic agent for many diseases caused by pathogenic microorganisms until early 1970. But, it carried serious side effects such as fever, eruption and pain and induced severe neurotoxicity and hepatotoxicity (Pedersen M. F. et al., 1971, Invest. Urol. 9: p. 234-237). So, it has been replaced with other antibiotics with improved stability and most recently it is only being applied on local wounds as a form of ointment.
According to the increased use of antibiotics, pathogenic microorganisms having resistance to those antibiotics have been frequently noticed. In the midst, polymyxin draws our attention since it has excellent bactericidal effect on Gram-negative bacteria, in particular Pseudomonas aeruginosa and Acinetobacter baumannii exhibiting resistance against β-lactam, aminoglycoside and fluoroquinolone antibiotics.
Levin, et al reported that colistin (polymyxin E) was intravenously injected to 60 patients infected with Pseudomonas aeruginosa and Acinetobacter baumannii exhibiting resistance against the conventional antibiotics and as a result 58% of the patients were improved (Levin A. S. et al. 1999. Clin. Infect. Dis. 28:1008-1011). And there is another report by Stein, et al. saying that 3 osteomyelitis patients infected with Pseudomonas aeruginosa having resistance against almost all antibiotics were improved by the treatment of colistin (Stein A. et al., 2002, Clin. Infect. Dis. 35: p. 901-902). In another report, meningitis caused by Acinetobacter having resistance against antibiotics was also successfully treated by colistin (Jimenez-Mejias M. E. et al., 2002. Eur. J. Clin. Microbiol. Infect. Dis. 21: p. 212-214). Another report says that ventriculis caused by antibiotics-resistant Klebsiella pneumoniae was successfully treated by polymyxin B (Segal-Maurer S. et al., 1999, Clin. Infect. Dis. 28: p. 1134-1138).
As described hereinbefore, polymyxin seems to have therapeutic effect on Gram-negative bacteria having resistance against the conventional antibiotics, so that it is in increasing demand.
It had been tried to introduce an antibiotic biosynthesis gene into an industrially mass-productive strain in order to increase antibiotic productivity (Eppelmann K. et al., 2001. J. Biol. Chem. 276: p. 34824-34831; Pfeifer B. A. et al., 2001, Microbiol. Mol. Biol. Rev. 65: 106-118) and in fact it was confirmed that the substitution of a promoter of the antibiotic biosynthesis gene with a stronger one resulted in the increase of productivity (Tsuge K. et al., 2001. J. Bacteriol. 183: p. 6265-6273). There is an attempt to develop a novel antibiotic by re-constructing modules or domains of an antibiotic biosynthesis gene (Mootz H. D. et al., 2000. Proc. Natl. Acad. Sci. USA 97: p. 5848-5853; Ferra F. D. et al., 1997. J. Biol. Chem. 272: p. 25304-25309) or substituting a specific amino acid of the domains (Eppelmann K. et al., Biochemistry 41: p. 9718-9726). However, no polymyxin biosynthesis gene has been identified so far, therefore it had hardly been tried to increase productivity or develop a novel antibiotic based on the above mentioned techniques.
Therefore, it is important to identify a polymyxin biosynthesis gene and secure the information on the gene to increase production of polymyxin or develop polymyxin with less side effects and polymyxin based novel antibiotics.
The present inventors isolated, purified and analyzed polymyxin from Paenibacillus polymyxa E681. And the inventors confirmed that the strain produced polymyxin and found out and isolated a gene cluster encoding NRPS by sequencing the entire nucleotide sequence. The present inventors finally completed this invention by confirming with the domain analysis that the gene cluster was polymyxin biosynthesis gene.